ROMK K+ channels play an essential role in K+ secretion in cortical collecting ducts (CCDs). The activity of the ROMK channels are regulated by multiple signaling pathways including protein kinase A (PKA), protein kinase C (PKC) and intracellular pH (pHi). Recently, we reported a novel mechanism for regulation of ROMK via direct interaction with membrane phospholipid, phosphatidylinositol-4 ,5-bisphosphate (PIP2). The interaction occurs between positively charged amino acids in the proximal C-terminal region of ROMK1 and PIP2. We hypothesize that the direct interaction between the anionic PIP2 in the inner leaflet of the plasma membrane and the cationic amino acids in this proximal C-terminal region of ROMK1 stabilizes the channels in the open state. The long-term objectives of PI's research are to understand the molecular mechanisms for PIP2 regulation of ROMK and the physiological importance of this regulation. First, we will test the hypothesis that PIP2 regulates ROMK1 opening by anchoring the proximal C-terminal region of the channel to the plasma membrane. Binding of green fluorescent protein (GFP) fusion proteins of the C-terminus of ROMK to plasma membrane PIP2 in living cells will be examined using laser scanning confocal imaging system. Effects of anchoring the proximal C-terminus of ROMK1 to the plasma membrane on channel activity will be studied using palmitoylation as an alternative membrane anchor. Second, we will examine the molecular mechanism for regulation of the K+ channels in CCDs by PLC-activating hormones. Activation of PKC by phorbol ester inhibits K+ channels in rat CCDs. This effect of PKC is likely the mechanism for regulation of K+ secretion by the phospholipase C (PLC)-activating hormones such as bradykinin and epidermal growth factor. Our preliminary results showed that PKC inhibits K+ channels by reducing membrane PIP2 content. The effects of PKC on PIP2 content and on phosphorylation of ROMK in rat CCDs as well as in heterologous expression systems (such as cultured cells and Xenopus oocytes) will be studied. The biochemical studies will be correlated with electrophysiological recording of channel activity.